Intermediate-range order governs dynamics in dense colloidal liquids.

The conventional wisdom is that liquids are completely disordered and lack nontrivial structure beyond nearest-neighbor distances. Recent observations have upended this view and demonstrated that the microstructure in liquids is surprisingly rich and plays a critical role in numerous physical, biological, and industrial processes. However, approaches to uncover this structure are either system-specific or yield results that are not physically intuitive. Here, through single-particle resolved three-dimensional confocal microscope imaging and the use of a recently introduced four-point correlation function, we show that bidisperse colloidal liquids have a highly nontrivial structure comprising alternating layers with icosahedral and dodecahedral order, which extends well beyond nearest-neighbor distances and grows with supercooling. By quantifying the dynamics of the system on the particle level, we establish that it is this intermediate-range order, and not the short-range order, which has a one-to-one correlation with dynamical heterogeneities, a property directly related to the relaxation dynamics of glassy liquids. Our experimental findings provide a direct and much sought-after link between the structure and dynamics of liquids and pave the way for probing the consequences of this intermediate-range order in other liquid state processes.

The reversible activation of norovirus by metal ions.

Murine norovirus (MNV) undergoes extremely large conformational changes in response to the environment. The T = 3 icosahedral capsid is composed of 180 copies of ~58-kDa VP1 comprised of N-terminus (N), shell (S), and C-terminal protruding (P) domains. At neutral pH, the P domains are loosely tethered to the shell and float ~15 Å above the surface. At low pH or in the presence of bile salts, the P domain drops onto the shell and this movement is accompanied by conformational changes within the P domain that enhance receptor interactions while blocking antibody binding. While previous crystallographic studies identified metal binding sites in the isolated P domain, the ~2.7-Å cryo-electron microscopy structures of MNV in the presence of Mg2+ or Ca2+ presented here show that metal ions can recapitulate the contraction observed at low pH or in the presence of bile. Further, we show that these conformational changes are reversed by dialysis against EDTA. As observed in the P domain crystal structures, metal ions bind to and contract the G'H' loop. This movement is correlated with the lifting of the C'D' loop and rotation of the P domain dimers about each other, exposing the bile salt binding pocket. Isothermal titration calorimetry experiments presented here demonstrate that the activation signals (bile salts, low pH, and metal ions) act in a synergistic manner that, individually, all result in the same activated structure. We present a model whereby these reversible conformational changes represent a uniquely dynamic and tissue-specific structural adaptation to the in vivo environment.IMPORTANCEThe highly mobile protruding domains on the calicivirus capsids are recognized by cell receptor(s) and antibodies. At neutral pH, they float ~15 Å above the shell but at low pH or in the presence of bile salts, they contract onto the surface. Concomitantly, changes within the P domain block antibody binding while enhancing receptor binding. While we previously demonstrated that metals also block antibody binding, it was unknown whether they might also cause similar conformational changes in the virion. Here, we present the near atomic cryo-electron microscopy structures of infectious murine norovirus (MNV) in the presence of calcium or magnesium ions. The metal ions reversibly induce the same P domain contraction as low pH and bile salts and act in a synergistic manner with the other stimuli. We propose that, unlike most other viruses, MNV facilely changes conformations as a unique means to escape immune surveillance as it moves through various tissues.

Structural and antigenic characterization of the avian adeno-associated virus capsid.

IMPORTANCE: AAVs are extensively studied as promising therapeutic gene delivery vectors. In order to circumvent pre-existing antibodies targeting primate-based AAV capsids, the AAAV capsid was evaluated as an alternative to primate-based therapeutic vectors. Despite the high sequence diversity, the AAAV capsid was found to bind to a common glycan receptor, terminal galactose, which is also utilized by other AAVs already being utilized in gene therapy trials. However, contrary to the initial hypothesis, AAAV was recognized by approximately 30% of human sera tested. Structural and sequence comparisons point to conserved epitopes in the fivefold region of the capsid as the reason determinant for the observed cross-reactivity.

A review: analysis of technical challenges in cultured meat production and its commercialization.

The cultured meat technology has developed rapidly in recent years, but there are still many technical challenges that hinder the large-scale production and commercialization of cultured meat. Firstly, it is necessary to lay the foundation for cultured meat production by obtaining seed cells and maintaining stable cell functions. Next, technologies such as bioreactors are used to expand the scale of cell culture, and three-dimensional culture technologies such as scaffold culture or 3D printing are used to construct the three-dimensional structure of cultured meat. At the same time, it can reduce production costs by developing serum-free medium suitable for cultured meat. Finally, the edible quality of cultured meat is improved by evaluating food safety and sensory flavor, and combining ethical and consumer acceptability issues. Therefore, this review fully demonstrates the current development status and existing technical challenges of the cultured meat production technology with regard to the key points described above, in order to provide research ideas for the industrial production of cultured meat.

Heterogeneity of cancer stem cell-related marker expression is associated with three-dimensional structures in malignant pleural effusion produced by lung adenocarcinoma.

INTRODUCTION: Cancer stem cells have been described in lung adenocarcinoma-associated malignant pleural effusion. They show clinically important features, including the ability to initiate new tumours and resistance to treatments. However, their correlation with the three-dimensional tumour structures in the effusion is not well understood. METHODS: Cell blocks produced from lung adenocarcinoma patients' pleural effusion were examined for cancer stem cell-related markers Nanog and CD133 using immunocytochemistry. The three-dimensional cancer cell structures and CD133 expression patterns were visualized with tissue-clearing technology. The expression patterns were correlated with tumour cell structures, genetic variants and clinical outcomes. RESULTS: Thirty-nine patients were analysed. Moderate-to-strong Nanog expression was detected in 27 cases (69%), while CD133 was expressed by more than 1% of cancer cells in 11 cases (28%). Nanog expression was more homogenous within individual specimens, while CD133 expression was detected in single tumour cells or cells within small clusters instead of larger structures in 8 of the 11 positive cases (73%). Although no statistically significant correlation between the markers and tumour genetic variants or patient survival was observed, we recorded seven cases with follow-up specimens after cancer treatment, and four (57%) showed a change in stem cell-related marker expression corresponding to treatment response. CONCLUSIONS: Lung adenocarcinoma cells in the pleural effusion show variable expression of cancer stem cell-related markers, some showing a correlation with the size of cell clusters. Their expression level is potentially correlated with cancer treatment effects.

Structural Catalytic Core of the Members of the Superfamily of Acid Proteases.

The superfamily of acid proteases has two catalytic aspartates for proteolysis of their peptide substrates. Here, we show a minimal structural scaffold, the structural catalytic core (SCC), which is conserved within each family of acid proteases, but varies between families, and thus can serve as a structural marker of four individual protease families. The SCC is a dimer of several structural blocks, such as the DD-link, D-loop, and G-loop, around two catalytic aspartates in each protease subunit or an individual chain. A dimer made of two (D-loop + DD-link) structural elements makes a DD-zone, and the D-loop + G-loop combination makes a psi-loop. These structural markers are useful for protein comparison, structure identification, protein family separation, and protein engineering.

High-Risk Mucosal Human Papillomavirus 16 (HPV16) E6 Protein and Cutaneous HPV5 and HPV8 E6 Proteins Employ Distinct Strategies To Interfere with Interferon Regulatory Factor 3-Mediated Beta Interferon Expression.

Persistent infection with some mucosal α-genus human papillomaviruses (HPVs; the most prevalent one being HPV16) can induce cervical carcinoma, anogenital cancers, and a subset of head and neck squamous cell carcinoma (HNSCC). Cutaneous ß-genus HPVs (such as HPV5 and HPV8) associate with skin lesions that can progress into squamous cell carcinoma with sun exposure in Epidermodysplasia verruciformis patients and immunosuppressed patients. Here, we analyzed mechanisms used by E6 proteins from the α- and ß-genus to inhibit the interferon-ß (IFNB1) response. HPV16 E6 mediates this effect by a strong direct interaction with interferon regulatory factor 3 (IRF3). The binding site of E6 was localized within a flexible linker between the DNA-binding domain and the IRF-activation domain of IRF3 containing an LxxLL motif. The crystallographic structure of the complex between HPV16 E6 and the LxxLL motif of IRF3 was solved and compared with the structure of HPV16 E6 interacting with the LxxLL motif of the ubiquitin ligase E6AP. In contrast, cutaneous HPV5 and HPV8 E6 proteins bind to the IRF3-binding domain (IBiD) of the CREB-binding protein (CBP), a key transcriptional coactivator in IRF3-mediated IFN-ß expression. IMPORTANCE Persistent HPV infections can be associated with the development of several cancers. The ability to persist depends on the ability of the virus to escape the host immune system. The type I interferon (IFN) system is the first-line antiviral defense strategy. HPVs carry early proteins that can block the activation of IFN-I. Among mucosal α-genus HPV types, the HPV16 E6 protein has a remarkable property to strongly interact with the transcription factor IRF3. Instead, cutaneous HPV5 and HPV8 E6 proteins bind to the IRF3 cofactor CBP. These results highlight the versatility of E6 proteins to interact with different cellular targets. The interaction between the HPV16 E6 protein and IRF3 might contribute to the higher prevalence of HPV16 than that of other high-risk mucosal HPV types in HPV-associated cancers.

Isolation and protein MdtQ analysis of outer membrane vesicles released by carbapenem-resistant Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged as a leading public health problem, and is increasingly being reported worldwide with resistance to a wide spectrum of antibiotics. Recent reports have demonstrated that the outer membrane vesicles (OMVs) of gram-negative bacteria are potent resistance factors, but their role in the drug resistance of CRKP has not been elucidated. In order to investigate the effects of OMV components on drug resistance and to explore the mechanism of antimicrobial resistance in CRKP, we isolated the OMVs through ultracentrifugation, separated the OMV proteins through mass spectrometry (MS), and performed bioinformatics analysis. A total of 3,192 proteins were detected by nano LC-MS/MS analysis, with 108 (61.4%) cytoplasmic proteins, 50 (28.4%) cytoplasmic membrane proteins, nine (5.1%) periplasmic proteins, six (3.4%) outer membrane proteins, two (1.1%) extracellular proteins, and one (0.6%) other protein detected in the vesicles. MdtQ was detected as the only multidrug resistance outer membrane protein. Further experiments confirmed that MdtQ included the 1440 BP sequence and had a unique three-dimensional structure. To superimpose MdtQ with KPC-2 resistant proteins, I7ACB1, I7AKP2, and Q93LQ9, the root mean square deviation (RMSD) values were calculated (0.379, 0.671, and 1.35, respectively). I7ACB1 had the lowest RMSD value, indicating that it had the best superimposition effect. Furthermore, MdtQ had 20 biological pocket structures, and the four most important pockets were evenly distributed around the inner perimeter of its three-dimensional structure. These findings may provide a theoretical basis for controlling the spread of bacterial resistance in the future.

Synthesis, Structures, and Properties of π-Extended Phosphindolizine Derivatives.

Dibenzo[b,g]phosphindolizine oxide and three types of benzo[e]naphthophosphindolizine oxides have been synthesized by the ring-closing metathesis of benzo[b]phosphole oxide and naphthophosphole oxides with two olefin tethers. Their molecular structures and properties were revealed by X-ray crystallographic analysis, UV-vis spectroscopy, and electrochemical analysis. The number and position of the benzene rings were found to alter the structural geometry and the HOMO/LUMO energy levels, and their effects were investigated by theoretical calculations. Among the phosphindolizine oxide derivatives investigated, only benzo[e]naphtho[2,3-b]phosphindolizine oxide with the naphthalene ring fused at 2,3-positions showed weak yellow fluorescence with a large Stokes shift.

Lactoferricin, an antimicrobial motif derived from lactoferrin with food preservation potential.

The growth of bacteria and fungi may cause disease inf human or spoilage of food. New antimicrobial substances need to be discovered. Lactoferricin (LFcin) is a group of antimicrobial peptides derived from the N-terminal region of the milk protein lactoferrin (LF). LFcin has antimicrobial ability against a variety of microorganisms, which is significantly better than that of its parent version. Here, we review the sequences, structures, and antimicrobial activities of this family and elucidated the motifs of structural and functional significance, as well as its application in food. Using sequence and structural similarity searches, we identified 43 new LFcins from the mammalian LFs deposited in the protein databases, which are grouped into six families according to their origins (Primates, Rodentia, Artiodactyla, Perissodactyla, Pholidota, and Carnivora). This work expands the LFcin family and will facilitate further characterization of novel peptides with antimicrobial potential. Considering the antimicrobial effect of LFcin on foodborne pathogens, we describe the application of these peptides from the prospective of food preservation.

Characterisation of Elevenin-Vc1 from the Venom of Conus victoriae: A Structural Analogue of α-Conotoxins.

Elevenins are peptides found in a range of organisms, including arthropods, annelids, nematodes, and molluscs. They consist of 17 to 19 amino acid residues with a single conserved disulfide bond. The subject of this study, elevenin-Vc1, was first identified in the venom of the cone snail Conus victoriae (Gen. Comp. Endocrinol. 2017, 244, 11-18). Although numerous elevenin sequences have been reported, their physiological function is unclear, and no structural information is available. Upon intracranial injection in mice, elevenin-Vc1 induced hyperactivity at doses of 5 or 10 nmol. The structure of elevenin-Vc1, determined using nuclear magnetic resonance spectroscopy, consists of a short helix and a bend region stabilised by the single disulfide bond. The elevenin-Vc1 structural fold is similar to that of α-conotoxins such as α-RgIA and α-ImI, which are also found in the venoms of cone snails and are antagonists at specific subtypes of nicotinic acetylcholine receptors (nAChRs). In an attempt to mimic the functional motif, Asp-Pro-Arg, of α-RgIA and α-ImI, we synthesised an analogue, designated elevenin-Vc1-DPR. However, neither elevenin-Vc1 nor the analogue was active at six different human nAChR subtypes (α1ß1εδ, α3ß2, α3ß4, α4ß2, α7, and α9α10) at 1 µM concentrations.

Revealing the three-dimensional structure of liquids using four-point correlation functions.

Disordered systems like liquids, gels, glasses, or granular materials are not only ubiquitous in daily life and in industrial applications, but they are also crucial for the mechanical stability of cells or the transport of chemical and biological agents in living organisms. Despite the importance of these systems, their microscopic structure is understood only on a rudimentary level, thus in stark contrast to the case of gases and crystals. Since scattering experiments and analytical calculations usually give only structural information that is spherically averaged, the three-dimensional (3D) structure of disordered systems is basically unknown. Here, we introduce a simple method that allows probing of the 3D structure of such systems. Using computer simulations, we find that hard sphere-like liquids have on intermediate and large scales a simple structural order given by alternating layers with icosahedral and dodecahedral symmetries, while open network liquids like silica have a structural order with tetrahedral symmetry. These results show that liquids have a highly nontrivial 3D structure and that this structural information is encoded in nonstandard correlation functions.

Progress of Dispersants for Coal Water Slurry.

Dispersants, serving as an essential raw material in the formulation of coal water slurry, offer an economical and convenient solution for enhancing slurry concentration, thus stimulating significant interest in the development of novel and efficient dispersants. This paper intends to illuminate the evolution of dispersants by examining both the traditional and the newly conceived types and elaborating on their respective mechanisms of influence on slurry performance. Dispersants can be classified into anionic, cationic, amphoteric, and non-ionic types based on their dissociation properties. They can be produced by modifying either natural or synthetic products. The molecular structure of a dispersant allows for further categorization into one-dimensional, two-dimensional, or three-dimensional structure dispersants. This document succinctly outlines dispersants derived from natural products, three-dimensional structure dispersants, common anionic dispersants such as lignin and naphthalene, and amphoteric and non-ionic dispersants. Subsequently, the adsorption mechanism of dispersants, governed by either electrostatic attraction or functional group effects, is elucidated. The three mechanisms through which dispersants alter the surface properties of coal, namely the wetting dispersion effect, electrostatic repulsion effect, and steric hindrance effect, are also explained. The paper concludes with an exploration of the challenges and emerging trends in the domain of dispersants.

Molecular dynamic (in silico) modeling of structure-function of glutelin type-B 5-like from proso millet storage protein: effects of temperature and pressure.

Molecular dynamic (MD) simulation provides an insight into the behavior of a protein under applied processing at the molecular level. The behavior of glutelin type-B 5-like protein, a type of glutelin protein from proso millet was studied, in solution under different temperatures (300, 350, and 400 K) and pressure (1 bar, 3 kbar, and 6 kbar) levels using a molecular dynamics simulation approach. The combined treatment effect (400 K, 6 kbar) increased the compaction of the protein compared to the level at (300 K, 1 bar) as shown by the decreased radius of gyration values from 3.26 to 2.92 nm, decreased solvent accessibility surface area from 327.47 to 311.06 nm2 and decreased volume from 108.35 to 105.04 nm3. The root means square deviation increased with increasing temperature but decreased with increasing pressure while the root means square fluctuations increased significantly with increased in temperature and pressure. A snapshot of the three-dimensional structure of the protein revealed compression of its occluded cavities at higher pressure levels but no obvious disruption to the secondary structure elements of the protein was observed, except for the loss of a few amino acid residues that comprise the secondary structure element. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05594-y.

A Genomic Island of Vibrio cholerae Encodes a Three-Component Cytotoxin with Monomer and Protomer Forms Structurally Similar to Alpha-Pore-Forming Toxins.

Alpha-pore-forming toxins (α-PFTs) are secreted by many species of bacteria, including Escherichia coli, Aeromonas hydrophila, and Bacillus thuringiensis, as part of their arsenal of virulence factors, and are often cytotoxic. In particular, for α-PFTs, the membrane-spanning channel they form is composed of hydrophobic α-helices. These toxins oligomerize at the surface of target cells and transition from a soluble to a protomer state in which they expose their hydrophobic regions and insert into the membrane to form a pore. The pores may be composed of homooligomers of one component or heterooligomers with two or three components, resulting in bi- or tripartite toxins. The multicomponent α-PFTs are often expressed from a single operon. Recently, motility-associated killing factor A (MakA), an α-PFT, was discovered in Vibrio cholerae. We report that makA is found on the V. cholerae GI-10 genomic island within an operon containing genes for two other potential α-PFTs, MakB and MakE. We determined the X-ray crystal structures for MakA, MakB, and MakE and demonstrated that all three are structurally related to the α-PFT family in the soluble state, and we modeled their protomer state based on the α-PFT AhlB from A. hydrophila. We found that MakA alone is cytotoxic at micromolar concentrations. However, combining MakA with MakB and MakE is cytotoxic at nanomolar concentrations, with specificity for J774 macrophage cells. Our data suggest that MakA, -B, and -E are α-PFTs that potentially act as a tripartite pore-forming toxin with specificity for phagocytic cells. IMPORTANCE The bacterium Vibrio cholerae causes gastrointestinal, wound, and skin infections. The motility-associated killing factor A (MakA) was recently shown to be cytotoxic against colon, prostate, and other cancer cells. However, at the outset of this study, the capacity of MakA to damage cells in combination with other Mak proteins encoded in the same operon had not been elucidated. We determined the structures of three Mak proteins and established that they are structurally related to the α-PFTs. Compared to MakA alone, the combination of all three toxins was more potent specifically in mouse macrophages. This study highlights the idea that the Mak toxins are selectively cytotoxic and thus may function as a tripartite toxin with cell type specificity.

Fungal effector SIB1 of Colletotrichum orbiculare has unique structural features and can suppress plant immunity in Nicotiana benthamiana.

Fungal plant pathogens secrete virulence-related proteins, called effectors, to establish host infection; however, the details are not fully understood yet. Functional screening of effector candidates using Agrobacterium-mediated transient expression assay in Nicotiana benthamiana identified two virulence-related effectors, named SIB1 and SIB2 (Suppression of Immunity in N. benthamiana), of an anthracnose fungus Colletotrichum orbiculare, which infects both cucurbits and N. benthamiana. The Agrobacterium-mediated transient expression of SIB1 or SIB2 increased the susceptibility of N. benthamiana to C. orbiculare, which suggested these effectors can suppress immune responses in N. benthamiana. The presence of SIB1 and SIB2 homologs was found to be limited to the genus Colletotrichum. SIB1 suppressed both (i) the generation of reactive oxygen species triggered by two different pathogen-associated molecular patterns, chitin and flg22, and (ii) the cell death response triggered by the Phytophthora infestans INF1 elicitin in N. benthamiana. We determined the NMR-based structure of SIB1 to obtain its structural insights. The three-dimensional structure of SIB1 comprises five ß-strands, each containing three disulfide bonds. The overall conformation was found to be a cylindrical shape, such as the well-known antiparallel ß-barrel structure. However, the ß-strands were found to display a unique topology, one pair of these ß-strands formed a parallel ß-sheet. These results suggest that the effector SIB1 present in Colletotrichum fungi has unique structural features and can suppress pathogen-associated molecular pattern-triggered immunity in N. benthamiana.

A Heat-Induced Mutation on VP1 of Foot-and-Mouth Disease Virus Serotype O Enhanced Capsid Stability and Immunogenicity.

Foot-and-mouth disease (FMD) is a highly contagious viral disease affecting cloven-hoofed animals that causes a significant economic burden globally. Vaccination is the most effective FMD control strategy. However, FMD virus (FMDV) particles are prone to dissociate when appropriate physical or chemical conditions are unavailable, such as an incomplete cold chain. Such degraded vaccines result in compromised herd vaccination. Therefore, thermostable FMD particles are needed for use in vaccines. This study generated thermostable FMDV mutants (M3 and M10) by serial passages at high temperature, subsequent amplification, and purification. Both mutants contained an alanine-to-threonine mutation at position 13 in VP1 (A1013T), although M3 contained 3 additional mutations. The selected mutants showed improved stability and immunogenicity in neutralizing antibody titers, compared with the wild-type (wt) virus. The sequencing analysis and cryo-electron microscopy showed that the mutation of alanine to threonine at the 13th amino acid in the VP1 protein (A1013T) is critical for the capsid stability of FMDV. Virus-like particles containing A1013T (VLPA1013T) also showed significantly improved stability to heat treatment. This study demonstrated that Thr at the 13th amino acid of VP1 could stabilize the capsid of FMDV. Our findings will facilitate the development of a stable vaccine against FMDV serotype O. IMPORTANCE Foot-and-mouth disease (FMD) serotype O is one of the global epidemic serotypes and causes significant economic loss. Vaccination plays a key role in the prevention and control of FMD. However, the success of vaccination mainly depends on the quality of the vaccine. Here, the thermostable FMD virus (FMDV) mutants (M3 and M10) were selected through thermal screening at high temperatures with improved stability and immunogenicity compared with the wild-type virus. The results of multisequence alignment and cryo-electron microscopy (cryo-EM) analysis showed that the Thr substitution at the 13th amino acid in the VP1 protein is critical for the capsid stability of FMDV. For thermolabile type O FMDV, this major discovery will aid the development of its thermostable vaccine.

Contact networks in RNA: a structural bioinformatics study with a new tool.

Base pairing in RNA are significantly rich and versatile due to the potential non-canonical base pairing amongst nucleotides. Not only that, one base in RNA can pair with more than one bases simultaneously. This opens up a new dimension of research to detect such types of base-base pair networks in RNA and to analyze them. Even if a base do not form a pair, it may have significant extent of [Formula: see text]-[Formula: see text] stacking overlap that can stabilize the structures. In this work, we report a software tool, called BPNet, that accepts a mmCIF or PDB file and computes the base-pair/[Formula: see text]-[Formula: see text] contact network components using graph formalism. The software can run on Linux platform in both serial and parallel modes. It generates several information in suitable file formats for visualization of the networks. This paper describes the BPNet software and also presents some interesting results obtained by analyzing several RNA structures by the software to show its effectiveness.

Diversity of amino acid substitutions of penicillin-binding proteins in penicillin-non-susceptible and non-vaccine type Streptococcus pneumoniae.

PURPOSE: In Japan, the introduction of pneumococcal conjugate vaccine (PCV) in children has decreased vaccine-type (VT) pneumococcal infections caused by penicillin (PEN)-non-susceptible Streptococcus pneumoniae. PEN-non-susceptible strains have gradually emerged among non-vaccine types (NVT). In this study, we aim to investigate the pbp gene mutations and the characteristics of PEN-binding proteins (PBPs) that mediate PEN resistance in NVT strains. MATERIALS AND METHODS: Pneumococcal 41 strains of NVT isolated from patients with invasive pneumococcal infection were randomly selected. Nucleotide sequences for pbp genes encoding PBP1A, PBP2X, and PBP2B were analyzed, and amino acid (AA) substitutions that contribute to ß-lactam resistance were identified. In addition, the three-dimensional (3D) structure of abnormal PBPs in the resistant strain was compared with that of a reference R6 strain via homology modeling. RESULTS: In PEN-non-susceptible NVT strains, Thr to Ala or Ser substitutions in the conserved AA motif (STMK) were important in PBP1A and PBP2X. In PBP2B, substitutions from Thr to Ala, adjacent to the SSN motif, and from Glu to Gly were essential. The 3D structure modeling indicated that AA substitutions are characterized by accumulation around the enzymatic active pocket in PBPs. Many AA substitutions detected throughout the PBP domains were not associated with resistance, except for AA substitutions in or adjacent to AA motifs. Clonal complexes and sequence types showed that almost all NVT cases originated in other countries and spread to Japan via repeat mutations. CONCLUSIONS: NVT with diverse AA substitutions increased gradually with pressure from both antimicrobial agents and vaccines.

Distinct structural transitions of chromatin topological domains correlate with coordinated hormone-induced gene regulation.

The human genome is segmented into topologically associating domains (TADs), but the role of this conserved organization during transient changes in gene expression is not known. Here we describe the distribution of progestin-induced chromatin modifications and changes in transcriptional activity over TADs in T47D breast cancer cells. Using ChIP-seq (chromatin immunoprecipitation combined with high-throughput sequencing), Hi-C (chromosome capture followed by high-throughput sequencing), and three-dimensional (3D) modeling techniques, we found that the borders of the ∼ 2000 TADs in these cells are largely maintained after hormone treatment and that up to 20% of the TADs could be considered as discrete regulatory units where the majority of the genes are either transcriptionally activated or repressed in a coordinated fashion. The epigenetic signatures of the TADs are homogeneously modified by hormones in correlation with the transcriptional changes. Hormone-induced changes in gene activity and chromatin remodeling are accompanied by differential structural changes for activated and repressed TADs, as reflected by specific and opposite changes in the strength of intra-TAD interactions within responsive TADs. Indeed, 3D modeling of the Hi-C data suggested that the structure of TADs was modified upon treatment. The differential responses of TADs to progestins and estrogens suggest that TADs could function as "regulons" to enable spatially proximal genes to be coordinately transcribed in response to hormones.